Unraveling the Structure of a Single DNA During Overstretching Using Multicolor Fluorescence Imaging

Abstract

Single-molecule manipulation techniques have provided a comprehensive understanding of the elastic behavior of DNA. We now know that its double-helical structure yields a significant persistence against bending on length scales of 150 base pairs and smaller. Interestingly, optical tweezers studies have also revealed, that at 65pN DNA undergoes a phase transition to another structure with significantly different elastic properties. Over a very narrow force range the polymer gains ∼70% in contour length, and becomes significantly more flexible.Until now the basic microscopic structure of overstretched DNA is under debate. Two qualitatively different models disagree on the molecular mechanism of the overstretching transition. The first one suggests that the DNA double helix unwinds to form a new structure, named S-DNA, which is usually depicted as a ladder with intact base pairing. The second model states that DNA overstretching is a force-induced melting transition, in which the hydrogen bonds between the two strands gradually break to yield single-stranded DNA, similar to thermal melting.Using a combination of fluorescence microscopy and optical tweezers we directly visualize the DNA overstretching transition and demonstrate that it is driven by melting of the double-stranded DNA. In the experiments we use intercalating dyes and fluorescently labeled single-stranded binding proteins to specifically visualize double- and single-stranded segments in DNA molecules undergoing the transition. Our data unambiguously show that the overstretching transition comprises a gradual conversion from double-stranded to single-stranded DNA, in agreement with the force-induced melting model. Interestingly, not predicted by either model, we found that melting is nucleation-limited, typically initiating from DNA extremities and nicks.